Protease has long been used in industry, and has found utility in a diversity of fields, including detergents such as laundry detergents, fiber modifying agents, leather processing agents, cosmetic compositions, bath additives, food-modifying agents, and pharmaceuticals. Of these, proteases for detergent use are produced in the largest amounts on an industrial scale. Examples of such known proteases that are derived from bacillus include ALCALASE, SAVINASE (registered trademarks; Novozymes), MAXACAL (registered trademark; Genencor), BLAP (registered trademark; Henkel), and KAP (Kao Corporation).
The purpose of incorporating protease into a detergent is to degrade protein soil adhering to clothes. Such soil actually is a “complex” soil formed of a plurality of organic and inorganic components, including not only proteins but also lipids originating from sebum, solid particles, and other substances. Therefore, demand has arisen for a detergent having excellent detergency against such complex soil.
Under the above situation, some of the present inventors have previously discovered several species of alkaline protease which have a molecular weight of about 43,000, exhibit a sufficient casein-degrading activity even in the presence of a fatty acid at a high concentration, and also exhibit excellent detergency not only to proteins but also to complex soils which include sebum and other substances, and filed a patent application therefor (see Patent Publication WO99/18218). Since the discovered alkaline proteases differ from subtilisin (which is a conventionally known serine protease derived from a microorganism belonging to the genus Bacillus) in terms of molecular weight, primary structure, enzymological characteristics and resistance to oxidants (the alkaline proteases are strongly resistant to oxidants) their classification into a new subtilisin subfamily has been proposed (Saeki et al., Biochem. Biophys. Res. Commun., 279, 313-319, 2000).
Incorporating such proteases into a detergent requires several steps, including concentration of a culture, drying, and granulation. Also, deactivation of proteases, which may otherwise occur during storage of the detergent, must be prevented.
In addition, researchers have recognized the problem that when a gene coding for such a protease is modified to give mutants having an enhanced specific activity and high production, some of such mutants show thermal stability lower than that of a protease before undergoing mutation. Thus, there has arisen a demand to improve thermal stability of the enzymes to solve the mentioned problems.
Accordingly, the present invention provides an alkaline protease which has excellent detergency against complex soil as well as enhanced thermal stability.